The ability of myoblasts to migrate through connective tissue barriers has important implications for muscle development, muscle regeneration, and myoblast-mediated gene transfer. During embryonic development, myogenic precursor cells migrate out of the somites and into the developing limb buds to form the limb musculature (Christ et al. "Experimental analysis of the origin of the wing musculature in avian embryos" Anat. Embrylo. 150:171-186, 1977), and myoblasts retain the ability to traverse the myofiber basal lamina during postnatal development (Hughes and Blau "Migration of myoblasts across basal lamina during skeletal muscle development" Nature 345:350-352, 1990). A number of studies have also demonstrated migration of myoblasts both within (Schultz et al. "Absence of exogenous satellite cell contribution to regeneration of frozen skeletal muscle" J. Muscle Res. Cell Motil 7:361-367, 1986; Philips et al. "Migration of myogenic cells in the rat extensor digitorum longus muscle studied with a split autograft model" Cell Tissue Res 262:81-88, 1990) and between adult muscles (Watt et al. "The movement of muscle precursor cells between adjacent regenerating muscles in the mouse" Anat. Embryol. 175:527-536, 1987; Watt et al. "Migration of LacZ positive cells from the tibialis anterior to the extensor digitorum longus muscle of the X-linked muscular dystrophic (MDX) mouse" J. Muscle Res. Cell Motil. 14:121-132, 1993; Watt et al. "Migration of muscle cells" Nature 368:496-407, 1994; Moens et al. "Lack of myoblast migration between transplanted and host muscle of mdx and normal mice" J. Muscle Res. Cell Motil. 17:37-43, 1996). These studies have shown that in order to produce myoblast migration between muscles there must first be disruption of the thick outer epimysium on one or both muscles, combined with some sort of chemotactic stimulus or stimuli generated by conditions such as inflammation or regeneration of muscle.
In recent years, myoblast cell therapy and myoblast-mediated gene transfer therapy have been extensively explored for both muscle disorders, such as muscular dystrophy (Karpati et al. "Myoblast transfer in Duchenme muscular dystrophy" Ann. Neurol 34:8-17, 1993; Morgan et al. "Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse" J. Cell. Biol 111:2437-2449, 1990), and for disorders which require production of systemic protein factors such as factor IX (Yao and Kurachi "Expression of human factor IX in mice after injection of genetically modified myoblasts" Proc. Natl. Acad. Sci USA 89:3357-3361, 1992; Roman et al. "Circulating human or canine factor IX from retrovirally transduced primary myoblasts and established myoblast cell lines grafted into murine skeletal muscle" Somatic Cell Mol. Genetics 18:247-258, 1992; Yao et al. "Primary myoblast-mediated gene transfer: persistent expression of human factor IX in mice" Gene Therapy 1:99-107, 1994; Wane, et al. "Persistent systematic production of human factor IX in mice by skeletal myoblast-mediated gene transfer: feasibility of repeat application to obtain therapeutic levels" Blood 90:1075-1082, 1997). The implanted myoblasts not only fuse with the existing myofibers, but can also remain as satellite cells (Yao and Kurachi "Implanted myoblasts not only fuse with myofibers but also survive as muscle precursor cells" J. Cell Sci. 105:957-963, 1993), but in both cases these myoblasts must traverse the basal lamina. However, the results from clinical trials using myoblast cell therapy for Duchenne's muscular dystrophy (DMD) have been equivocal, with some reporting success (Law et al. "Human gene therapy with myoblast transfer" Transplant. Proc. 29:2234-2237, 1990; Huard et al. "Human myoblast transplantation: preliminary results of 4 cases" Muscle & Nerve 15:550-560, 1992) and others reporting less encouraging results (Karpati et al. "Myoblast transfer in Duchenne muscular dystrophy" Ann. Neural. 34:8-17, 1993; Mendell et al. "Myoblast transfer in the treatment of Duchenne's muscular dystrophy" New England J. Med. 333:832-838, 1995). It is evident from these studies that substantial improvements are needed before such therapies will become practical as a therapeutic intervention for human disorders.
One of the primary limiting factors in myoblast therapy is the overall efficiency of incorporation of myoblasts into the myofibers. Estimates have suggested that only 5-10% of the implanted myoblasts become incorporated and contribute to transgene expression (Gussoni et al. "The fate of individual myoblasts after transplantation into muscles of DMD patients" Nature Medicine 3:970-977, 1997; Wang et al "Persistent systemic production of human factor IX in mice by skeletal myoblast-mediated gene transfer: feasibility of repeat application to obtain therapeutic levels" Blood 90:1075-1082, 1997). Evidence from human clinical trials of myoblast implantation to correct DMD has suggested that even when the immune system is suppressed by cyclosporine treatment, myoblast incorporation into the host myofibers is still low, and only minimal long term effects were noted (Karpati et al. "Myoblast transfer in Duchenne muscular dystrophy" Ann. Neurol. 34:8-17, 1993). These studies suggested that another barrier to successful myoblast incorporation is the presence of connective tissue sheaths surrounding both fascicles and individual myofibers. Myoblasts must first traverse these barriers to access the myofiber surface in order to fuse with and incorporate into the myofiber syncytium. Moreover, human muscle contains thicker connective tissue sheaths than that of smaller organisms, and therefore this barrier may be even greater in humans than in experimental animal models such as mice. Thus the ability of myoblasts to cross connective tissue barriers may have a major effect on the overall efficiency of the gene transfer process. Recent studies have also demonstrated that the myofiber basal lamina is a significant barrier to viral-mediated in vivo gene transfer as well (Huard et al. "The basal lamina is a physical barrier to herpes simplex virus-mediated gene delivery to mature muscle fibers" J. Virol. 70:8117-8123, 1996).
Physical and chemical disruption of the basal lamina by damaging the muscle would allow implanted myoblasts to cross the basal lamina and merge with the concomitant regeneration program, regenerating the muscle fibers with a mosaic of endogenous and implanted myonuclei. Most studies on myoblast transfer in animal models have used either physical injury (Wernig et al. "Formation of new muscle fibers and tumors after injection of cultured myogenic cells" J. Neurocytol. 20:982-997, 1991; Morgan et al. "Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse" J. Cell Biol. 111:2437-2449, 1990) or myotoxic agents (Salminen et al. "Implantation of recombinant rat myocytes into adult skeletal muscle: a potential gene therapy" Human Gene Therapy 2:15-26, 1991; Bonham et al. "Prolonged expression of therapeutic levels of human granulocyte-stimulating factor in rats following gene transfer to skeletal muscle" Human Gene Therapy 7:1423-1429, 1996) to produce this effect. However, these approaches may be too harmful and destructive for gene therapy in patients, particularly those suffering from disorders such as DMD or hemophilia.
Therefore, what is needed is a less destructive method for delivering genetically engineered therapeutics to muscles in the body.